1. Field of the Invention
The present invention relates to a capillary electrophoretic apparatus comprising a capillary electrophoretic part electrophoresing a sample injected into an end of a capillary column and detection means detecting each component separated in the capillary column at an appropriate position of the capillary column, and inclusively, it relates to a method and an apparatus characterized in sample injection into a capillary column.
2. Description of the Prior Art
Besides having those that comprise a single capillary column, capillary electrophoretic apparatuses also have a multi-capillary electrophoretic apparatus comprising a multi-capillary array migration part including a plurality of capillary columns for injecting a plurality of samples one by one into the capillary columns and simultaneously electrophoresing the samples in all capillary columns and an optical measuring part irradiating the capillary columns with light in the multi-capillary array migration part for measuring absorbance by the samples on the irradiated portion or fluorescence from the samples.
The capillary columns include those charged and not charged with gels for migration.
A capillary electrophoretic apparatus is applied for the separation of protein, and sequence determination for DNA. When applied to sequence determination for DNA, the capillary electrophoretic apparatus employs Sanger's reaction, electrophoreses a DNA fragment sample prepared by labeling a primer or a terminator with a fluorescent material and detects fluorescence from the DNA fragment sample during migration for determining the base sequence.
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for sequence determination for DNA having long base sequence such as a human genome. As one method, a multi-capillary DNA sequencer prepared by arranging a plurality of capillary columns in place of that employing a flat plate type slab gel is proposed. With such a capillary column, a sample can not only be readily handled or injected but also migrated at a high speed and detected in high sensitivity, when compared to the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat However, the capillary column hardly causes such problems and can perform detection in high sensitivity with small band spreading even if performing high-speed migration with application of a high voltage.
In capillary electrophoresis, a sample is injected into a capillary column under pressure or with application of a voltage. A method of electrophoretically injecting a sample is widely employed in general due to the simplicity of the apparatus structure, easiness of operations and excellent controllability of parameters.
In relation to the method of injecting the sample into the capillary column, migrational separability in later migrational separation must not be deteriorated and sample injection must be simpler.
Whether the capillary column is charged with a gel or not, one end of the capillary column must be dipped in a prepared sample, the other end must be dipped in a buffer solution, and an electrode such as a platinum wire must be dipped in the vicinity of the end of the capillary column within the sample. In a structure holding the electrode in the vicinity of the end of each capillary column in electrophoretic sample injection, the electrode structure for sample injection becomes complicated in the case of a multi-capillary electrophoretic apparatus collectively arranging a plurality of capillary columns in the form of an array for simultaneously performing electrophoresis. After dipping the end of the capillary column in a sample contained in a sample injection container and performing injection by applying a voltage or the like, the end of the capillary column must be transferred into a reservoir containing a buffer solution for migration. Thus, operations between sample injection and the start of migration are troublesome, and it is convenient if the operations can be automated.
In both of an electrophoretic apparatus comprising a single capillary column and a multi-capillary electrophoretic apparatus, migrational separability may be deteriorated in capillary electrophoresis charged with a gel, depending on the sample injection method. In the case of electrophoretically injecting a sample, a prescribed high voltage is applied within several seconds when starting voltage application and the high voltage is zeroed within several seconds when finishing voltage application in a conventional voltage application method, as shown in FIG. 4. Referring to FIG. 4, the vertical axis shows the voltage (kV) and the horizontal axis shows the time (second). In this example, a voltage of 7.6 kV is applied for 30 seconds. However, if high voltage is applied to the capillary column, remarkable stress is applied to a gel located on an end of the capillary column due to heat generation or the like to hinder injection of the sample into the capillary column, leading to such bad influence that the number of bases separable in migrational separation is limited. Furthermore, if the high voltage is abruptly applied, the gel may be forced out from the end of the capillary column due to electroosmosis flow. If the forced-out gel is damaged, the migrational separation state is disadvantageously deteriorated.
In slab gel electrophoresis, a large molecule (referred to as a macromolecule) such as template DNA having a size unreceivable in a gel matrix does not enter the gel matrix part but remains on an inlet for the gel matrix part. Even if force moving the macromolecule by driving force of an electric field is applied to a separation part in slab gel electrophoresis, the separation part has sufficient resistance against this force due to its sufficient volume to hardly become a problem. Also, in capillary electrophoresis, a macromolecule does not enter a gel matrix part but remains on an inlet for the gel matrix part (this phenomenon is referred to as clogging). Capillary electrophoresis has the following features: 1) the volume of a separation carrier is extremely small, 2) the material for the separation carrier is not a strong one such as an acrylamide gel but a viscous polymer solution, and 3) electric field strength per unit length is large. Therefore, force moving the macromolecule by driving force of an electric field is large, and resistance against this force is small. Thus, occurrence of clogging results in deterioration of a separation pattern.
In DNA sequence determination, since a macromolecule in a sample is template DNA, in order to solve the aforementioned problem resulting from clogging, therefore, the template DNA must be removed before injecting the sample into a capillary column. It is possible to remove the template DNA by chemical pretreatment For example, when preparing a DNA fragment sample by the Sanger's method, an enzyme, antigen or antibody is bonded to a primer for separating the DNA fragment sample from a template by enzyme reaction or antigen-antibody reaction after preparing the sample. However, such chemical pretreatment is troublesome and requires much labor and time.